US5130298A - Stabilized compositions containing epidermal growth factor - Google Patents

Stabilized compositions containing epidermal growth factor Download PDF

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US5130298A
US5130298A US07/353,131 US35313189A US5130298A US 5130298 A US5130298 A US 5130298A US 35313189 A US35313189 A US 35313189A US 5130298 A US5130298 A US 5130298A
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egf
zinc
composition
range
degradation
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John K. Cini
Amy L. Finkenaur
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Ethicon Inc
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Ethicon Inc
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Assigned to ETHICON, INC., A CORP. OF OHIO reassignment ETHICON, INC., A CORP. OF OHIO ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: CINI, JOHN K., FINKENAUR, AMY L.
Priority to US07/353,131 priority Critical patent/US5130298A/en
Priority to GR900100334A priority patent/GR1000392B/el
Priority to AU54968/90A priority patent/AU630952B2/en
Priority to KR1019900006828A priority patent/KR900017580A/ko
Priority to CA002016659A priority patent/CA2016659C/en
Priority to EP90305185A priority patent/EP0398619B1/en
Priority to DE90305185T priority patent/DE69002795T2/de
Priority to DK90305185.2T priority patent/DK0398619T3/da
Priority to JP2123208A priority patent/JP2763815B2/ja
Priority to PT94037A priority patent/PT94037B/pt
Priority to ES90305185T priority patent/ES2058799T3/es
Publication of US5130298A publication Critical patent/US5130298A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/315—Zinc compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions

Definitions

  • the present invention concerns pharmaceutical compositions containing human epidermal growth factor (EGF) and methods for making and using such compositions.
  • the invention relates to such pharmaceutical compositions having increased stability as a result of being combined with a metal cation, such as zinc.
  • Human EGF (also known as urogastrone) is a 53 amino acid polypeptide growth factor that has mitogenic activity for a number of kinds of cells, including epithelial and mesenchymal cells. Variants of the human EGF polypeptide have been reported, such as the 52 amino acid gamma-urogastrone. EGF has been reported to be useful in increasing the rate of wound healing as a result of its mitogenic effect. EGF has also been reported as being useful for treating gastric ulcers. A review of EGF is provided by Carpenter et al., in "Epidermal Growth Factor, Its Receptor and Related Proteins," Experimental Cell Research, 164:1-10 (1986).
  • EGF EGF-like growth factor
  • HPLC high performance liquid chromatography
  • EGF EGF-like growth factor
  • degradation products resulting from the degradation of EGF or derivatives resulting from the chemical modification of EGF. It is believed that there are at least three such degradation products, some or all of which have reduced EGF biological activity. Incubation of EGF at 45° C. accelerates the formation of the degradation products normally found with long term storage at ambient temperature. Such degradation, and the associated loss of biological activity of EGF, is a disadvantage because it makes it impractical to store aqueous or solid preparations of EGF over extended periods of time.
  • the present invention provides a means for reducing the degradation of the EGF molecule and the resulting loss of biological activity.
  • the present invention provides a pharmaceutical composition comprising a pharmaceutically effective amount of human epidermal growth factor (EGF) and an amount of a pharmaceutically acceptable metal cation sufficient to prevent the degradation of said EGF.
  • the metal cation is the divalent zinc ion.
  • a crystalline EGF composition which comprises a salt of a complex of zinc and EGF which is stabilized against degradation and loss of biological activity.
  • the present invention further provides methods for stabilizing EGF by mixing the EGF with a suitable metal cation which is capable of preventing the degradation of the EGF.
  • FIG. 1 is a chromatogram which depicts the relationship of EGF Peaks C, D, X and Y after a solution of aged EGF was analyzed by reverse phase HPLC.
  • Human EGF refers to the EGF having that polypeptide sequence, or any substantial portion thereof, as set forth in Urdea, M. S. et al., Proc. Natl. Acad. Sci. U.S.A. 80:6461-6465 (1983). Human EGF also refers to any human EGF variants, such as gamma-urogastrone. EGF may be isolated from natural sources, produced using recombinant DNA techniques or prepared by chemical synthesis. It is contemplated that biologically active fragments, analogs or man-made chemically synthesized derivatives of EGF may be used in the present invention instead of the entire naturally occurring molecule, provided that such fragments, analogs or derivatives retain the biological activity of naturally occurring EGF.
  • EGF biological activity refers to the mitogenic activities for epithelial and mesenchymal cells and the inhibition of gastric acid secretion.
  • EGF includes the EGF produced by any of the aforementioned methods and any bioactive fragments, analogs or derivatives and related polypeptides thereof.
  • analog of EGF refers to any polypeptide having a substantially identical amino acid sequence to EGF in which one or more amino acids have been substituted with chemically similar amino acids.
  • analog shall also include any polypeptide which has one or more amino acids deleted from or added to the EGF polypeptide, but which still retains a substantial amino acid sequence homology to EGF.
  • a substantial sequence homology is any homology greater than 50 percent.
  • fragment of EGF refers to any shorter version of EGF having at least 10 amino acid residues and having the same bioactivity as EGF.
  • chemical derivative refers to any polypeptide derived from the naturally occurring EGF polypeptide in which one or more amino acids have been chemically derivatized synthetically by reaction of functional side groups of the amino acids (i.e. it is derived from the parent EGF molecule by one or more steps).
  • a “pharmaceutically effective amount” of EGF refers to that amount which provides a therapeutic effect in various administration regimens. For example, when used for wound healing purposes it is that amount which is necessary to enhance the rate of healing a wound.
  • the compositions of the present invention may be prepared containing amounts of EGF within the range of from about 0.01 to about 1,000 micrograms per ml of an aqueous formulation. Preferably, the concentration is in the range 1-500 micrograms per ml and more preferably in the range 1-100 micrograms per ml.
  • EGF degrades over time to form multiple species of the EGF molecule which are believed to be degradation products.
  • Degradation of EGF refers to the natural aging process whereby the molecular structure of the EGF molecule that is used as a starting material (e.g., the 53 amino acid form or a variant thereof) is either: (a) chemically modified to form an EGF variant as a result of a naturally occurring or environmentally induced chemical reaction such as isomerization, oxidation or deamidation; or (b) broken down or decomposed into smaller molecules.
  • Such degradation occurs naturally as a result of environmental factors such as light, which can cause photooxidation; changes in pH; changes in ionic strength; changes in temperature; and physical manipulation of the molecule.
  • FIG. 1 depicts a reverse phase HPLC chromatogram of EGF showing the native EGF, designated Peak D, along with the three degradation products, designated Peaks C, X and Y.
  • Stabilized EGF runs as a predominantly single peak (Peak D) on reverse phase HPLC.
  • the major aging products of EGF appear to be those represented by Peaks X and Y.
  • the present invention has been shown to reduce the formation of Peaks X and Y, thus contributing to the maintenance of a predominantly active single species of EGF, i.e., greater than 90% of the starting material remains unchanged.
  • the present methods may also be used to stabilize other proteins through the blocking of active amino acid residues with zinc binding.
  • Such other proteins include those in which amino acid residues within the protein structure act to stabilize a degradation product, such as an isomerized intermediate.
  • such other proteins include those which have similar acting amino acid domains to EGF, such as other growth factors, for example fibroblast growth factor.
  • the terms “zinc”, “zinc cation”, or “zinc ion” all refer to the divalent zinc ion.
  • the present invention is exemplified by the use of the zinc divalent cation, it is contemplated that other suitable cations may achieve the same effect.
  • Such suitable cations must be “pharmaceutically acceptable,” which means that they are non-toxic to humans and have no harmful or undesirable side effects when administered to humans, such as inflammation or immunological reaction.
  • Such suitable cations must not cause the degradation of EGF, but rather must be capable of preventing such degradation.
  • such cations must not cause or induce free radical formation.
  • the cations must not adversely effect the biological properties of EGF but rather maintain such properties.
  • a suitable cation is one that is pharmaceutically acceptable, does not cause free radical formation and has EGF degradation preventing properties and EGF biological activity maintaining properties. It is contemplated that any monovalent, divalent or trivalent cation having such properties is within the scope of the present invention.
  • Lanthanum (trivalent) was shown to form a crystalline precipitate with EGF. Cations of the following metals would not be suitable because they cause free radical formation: manganese, copper, iron and cobalt.
  • Other cations which may be suitable are those of magnesium, calcium, cadmium, nickel, tin, potassium and lithium.
  • zinc-EGF or “zinc-EGF complex” refers to a complex ion wherein zinc is coordinately bound to EGF.
  • the zinc-EGF complex In aqueous solutions, the zinc-EGF complex must be maintained within the pH range 4.0-7.0, preferably between 5.5 and 6.0. Outside this range the zinc-EGF complex dissociates into monomeric EGF and zinc cation. Thus, it is preferred that aqueous solutions of zinc-EGF are buffered to maintain such a pH range. Any buffer system that maintains the pH within the range 4.0-7.0, preferably 5.5-6.0, is suitable, provided that the counterion present in the buffer does not chelate zinc or otherwise cause it to precipitate from solution.
  • buffer systems which contain phosphate or carbonate are not suitable because they will cause zinc to precipitate.
  • Suitable buffer systems are those based on the following counterions: acetate, succinate, chloride, sulfate, tartrate, malate, maleate and the like.
  • a preferred buffer system is an acetate buffer system.
  • zinc acetate is added to an EGF solution in order to stabilize the EGF.
  • Zinc acetate has the dual function of providing zinc ions to bind to EGF and acetate counterions to maintain the pH within the preferred range.
  • a buffer must also be used.
  • EGF EGF-derived neurotrophic factor
  • 10-20 mM zinc is used for each 250 micrograms EGF/ml.
  • These amounts may be varied by the skilled practitioner depending on the amount of EGF to be stabilized or the amount of crystalline Zn-EGF that is desired. For example, increasing the EGF concentration would require increasing the amount of zinc proportionately.
  • Higher amounts of zinc, such as up to 50 mM for each 250 micrograms EGF/ml may also be used to ensure a complete reaction.
  • Lower amounts of zinc such as 5-10 mM, may also be used but all of the EGF may not be stabilized. It is within the capabilities of one skilled in the art to make such variations.
  • the present invention also provides a crystalline EGF composition which comprises a salt of a complex of zinc and EGF, which has the advantage of long term storability.
  • the cation of the zinc-EGF salt may be any cation that is conducive to salt formation of zinc-EGF, such as: sodium, potassium, lithium, calcium, ammonium, magnesium or barium.
  • the zinc-EGF complex will precipitate out of solution provided the pH is within the range 4.0-7.0. Outside this range the complex dissociates.
  • EGF must be dissociated in order for it to have a biological effect in vivo.
  • zinc-EGF in aqueous or crystalline form, may be applied directly to a wound or other biological surface in order to provide a slow release formulation for EGF.
  • the physiological pH of the body fluids i.e. about neutral pH, will cause the zinc-EGF to slowly dissociate to release monomeric EGF.
  • Crystalline zinc-EGF may be prepared in any number of ways.
  • a soluble zinc salt such as zinc acetate or zinc chloride
  • the pH is adjusted to be within the range 4.0-7.0, preferably 5.5-6.0. Crystalline zinc-EGF then precipitates out of solution at room temperature and without mixing.
  • EGF in any form, such as lyophilized form may be added to a buffered zinc ion solution.
  • the zinc-EGF crystals may be recovered in any manner known in the art.
  • the aqueous solution containing the crystals may be centrifuged in order to Pack the crystals. Then, the supernatant is poured off and the crystals filtered, washed and then dried.
  • the compositions of the present invention may be used to treat wounds so as to increase the rate of healing thereof.
  • the types of wounds that may be healed using the compositions of the present invention are those which result from any accidental or medical injury which causes epithelial damage, such as ophthalmic wounds, such as those which result from corneal ulcers, radialkeratotomy, corneal transplants, epikeratophakia and other surgically induced wounds in the eye; and cutaneous wounds such as burn wounds, donor site wounds from skin transplants and ulcers (cutaneous, decubitis, venous stasis and diabetic).
  • compositions of the present invention may be treated with dermatological conditions in which the skin has been damaged, such as psoriasis, sunburn and skin rashes.
  • the compositions may be applied to the wound site either topically or internally depending on the type of wound.
  • Methods for increasing the rate of healing a wound comprise applying or contacting the compositions of the present invention directly to the wound by topically administering the composition to a wound site.
  • the composition is permitted to remain in contact with the wound for a period of time sufficient to increase the rate of cell growth at the wound site.
  • Such methods include incorporating any composition of the present invention into any pharmaceutically acceptable controlled release composition such as a cream, gel, aerosol spray microcapsules, films or lyophilized foams or aqueous formulation or soaking a gauze dressing with an aqueous solution of the composition and then applying such formulations or dressings to the wound site.
  • compositions of the present invention are useful in eyedrop formulations, eye gels, eye creams, liposome or micell formulations, aqueous vehicles for soaking soaked gauze dressings, burn dressings, artificial skins, sutures and staple coatings, salves or creams, gel formulations, foams and the like. Additional materials, such as buffers, preservatives, tonicity adjusting agents, anti-oxidants, polymers for adjusting viscosity or for use as extenders, and excipients may be used in the compositions.
  • Such other materials include acetate or borate buffers; thimerosol, sorbic acid, methyl or propyl paraben and chlorobutanol preservatives; sodium chloride and/or sugars to adjust the tonicity; and excipients such a mannitol, lactose or sucrose.
  • the structural stability of EGF as a result of metal binding is a function of the stoichiometry of metal to EGF, pH and ionic strength of the medium.
  • Another important variable in the metal-protein complex formation is the dielectric constant (DIE) and the water activity on the surface of the protein. Neutral compounds have been shown to affect the water structure and the DIE in the medium. A decrease in the DIE should cause an increase in ionic interaction (e.g., metal-EGF complex), thereby enhancing the binding of the metal to EGF and increasing the stability.
  • a secondary effect of decreasing the DIE would be to increase intramolecular hydrogen bonding, which would also contribute to the EGF stability.
  • Examples of neutral compounds which are capable of reducing the DIE are as follows: monohydric and polyhydric alcohols, such as ethanol, isopropanol, mannitol, sorbitol, inositol, sucrose, lactose, glycerin, and the like; polyhydroxylic compounds, such as glycerol, polyethylene glycol, propylene glycol, polyoxmer (Pluronic F-68), povidone, hydroxymethyl (ethyl or propyl) cellulose, octoxynol-9, and the like; surfactants, such as polysorbate (Tweens), Brij, polyoxyethylene sorbitan monoester and triester, and the like; amino acids, such as glycine, leucine
  • compositions may also be combined with anti-bacterial compounds, such as sulfadiazine compounds and in particular silver sulfadiazine and zinc sulfadiazine.
  • anti-bacterial compounds such as sulfadiazine compounds and in particular silver sulfadiazine and zinc sulfadiazine.
  • a zinc sulfadiazine compound would have the dual purpose of providing antibacterial and stabilizing properties to the composition.
  • compositions of the present invention may be lyophilized in order to provide further stability of the EGF.
  • Methods of lyophilization are well known in the art.
  • Stable lyophilized formulations containing growth factors are described in copending and commonly assigned U.S. Ser. No. 098,817, the contents of which is hereby incorporated by reference into this disclosure.
  • EGF stability with zinc ion was tested at pH 5.5 to 6.5. Lyophilized EGF was reconstituted in 50 mM sodium acetate at PH 5.5. A solution of zinc chloride was then added to this EGF solution. Alternatively, a solution of zinc acetate or crystals of a zinc salt could have been added. The pH was maintained in the range 5.5 to 6.5 and zinc-EGF precipitated out of solution at room temperature.
  • HPLC results are set forth below in Table 1.
  • the zinc-EGF complex must be dissociated before HPLC analysis because the complex cannot be run on the HPLC column as is. Therefore, prior to HPLC, the pH of the samples to be analyzed was adjusted to 3.0. Alternatively, EDTA (a chelating agent) may be used to reverse crystallization. Reverse phase HPLC was conducted using a Vydac C-4 column (4.6 mm ⁇ 25 cm, 5 ⁇ m). The flow rate was 0.8 ml/min at 26° C. using a linear gradient of 26% to 32% acetonitrile (0.1% TFA) over 26 minutes.
  • EDTA a chelating agent
  • This experiment was designed to answer the stability of the zinc-EGF complex results from the exclusion of water by the large and insoluble complex or alternatively from the direct interaction of zinc ion with specific amino acids in the protein.
  • a zinc-EGF solution and control were prepared as in Example 1 and the samples were adjusted to a PH of 4.6. At this pH EGF naturally precipitates out of solution since it is at its isoelectric point.
  • Two sets of the precipitated EGF at pH 4.6 in sodium acetate buffer were incubated for 7 days at 46° C. One set had zinc ion present, the other did not.
  • the HPLC results are set forth in Table 2 below.
  • Table 2 shows that the zinc-EGF complex has a significant decrease in Peaks X and Y compared to the formulation containing EGF alone in precipitated form. This suggests that a direct interaction of zinc with EGF is important and that the zinc-EGF complex is stable in both aqueous and crystalline forms.
  • the biological activity of the zinc-EGF complex was tested in the receptor binding assay (RBA).
  • RBA receptor binding assay
  • This assay measures the binding of EGF to its receptor and the assay has a variability range of 15-30%. It is an accepted method of determining the biological activity of EGF.
  • the receptor binding assay method used was that of Savage et al., Analytical Biochem, 111. pages 195 et seq. (1981).
  • the RBA is also described and set forth in U.S. Pat. No. 4,717,717, the disclosure of which is incorporated by reference into this specification.
  • EGF at a concentration of 100 micrograms per ml was mixed with zinc ion resulting in precipitation. The precipitated material was split into two equal volumes.
  • EDTA was added to one sample at twice the molar level of the zinc ion. This resulted in a clear solution with no precipitation. The other sample was left unchanged in a precipitated form and the three samples were assayed on the RBA and the results are set forth in Table 3.
  • the buffer used in the RBA was a phosphate buffer at pH 7.4. At this pH, the Zn-EGF dissociated into its monomeric form and the zinc precipitated as zinc phosphate. EGF without zinc was treated as a control.
US07/353,131 1989-05-16 1989-05-16 Stabilized compositions containing epidermal growth factor Expired - Lifetime US5130298A (en)

Priority Applications (11)

Application Number Priority Date Filing Date Title
US07/353,131 US5130298A (en) 1989-05-16 1989-05-16 Stabilized compositions containing epidermal growth factor
GR900100334A GR1000392B (el) 1989-05-16 1990-05-07 Σταθεροποιημενες συνθεσεις περιεχουσες επιδερμικο παραγοντα αναπτυξης.
AU54968/90A AU630952B2 (en) 1989-05-16 1990-05-11 Stabilized compositions containing epidermal growth factor
KR1019900006828A KR900017580A (ko) 1989-05-16 1990-05-14 표피 성장 인자를 함유하는 안정화된 조성물
CA002016659A CA2016659C (en) 1989-05-16 1990-05-14 Stabilized compositions containing epidermal growth factor
DE90305185T DE69002795T2 (de) 1989-05-16 1990-05-15 Stabilisierte, den epidermalen Wachstumsfaktor enthaltende Mittel.
EP90305185A EP0398619B1 (en) 1989-05-16 1990-05-15 Stabilized compositions containing epidermal growth factor
DK90305185.2T DK0398619T3 (da) 1989-05-16 1990-05-15 Stabiliserede præparater indeholdende epidermal vækstfaktor
JP2123208A JP2763815B2 (ja) 1989-05-16 1990-05-15 表皮成長因子を含有する安定化された組成物
PT94037A PT94037B (pt) 1989-05-16 1990-05-15 Processo para a preparacao de factor de crescimento epidermico (epidermal growth factor) e de composicoes farmaceuticas que o contem
ES90305185T ES2058799T3 (es) 1989-05-16 1990-05-15 Composiciones estabilizadas que contienen factor de crecimiento epidermico.

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EP (1) EP0398619B1 (el)
JP (1) JP2763815B2 (el)
KR (1) KR900017580A (el)
AU (1) AU630952B2 (el)
CA (1) CA2016659C (el)
DE (1) DE69002795T2 (el)
DK (1) DK0398619T3 (el)
ES (1) ES2058799T3 (el)
GR (1) GR1000392B (el)
PT (1) PT94037B (el)

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GR1000392B (el) 1992-06-30
GR900100334A (en) 1991-10-10
DE69002795D1 (de) 1993-09-23
EP0398619A3 (en) 1991-04-03
DE69002795T2 (de) 1994-01-13
ES2058799T3 (es) 1994-11-01
JP2763815B2 (ja) 1998-06-11
EP0398619A2 (en) 1990-11-22
JPH0334992A (ja) 1991-02-14
PT94037B (pt) 1996-11-29
CA2016659A1 (en) 1990-11-16
PT94037A (pt) 1991-01-08
AU630952B2 (en) 1992-11-12
KR900017580A (ko) 1990-12-19
CA2016659C (en) 1996-12-10
DK0398619T3 (da) 1993-10-04
EP0398619B1 (en) 1993-08-18
AU5496890A (en) 1990-11-22

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